The objective of the proposed study is to elucidate the mechanisms of basal and regulated transcription by RNA polymerase II. At present, most of the basal transcription factors have been purified and clone, and thus, the next several years should be a productive period for the analysis of the mechanism of the reconstituted with purified, recombinant TFII, TBP, and TFIIF30 along with purified RNA polymerase II from Drosophila, calf thymus, or HeLa cells. This minimal transcription system appears to constitute the central catalytic core of the RNA polymerase II transcriptional machinery. Beginning with this purified, minimal system, it will be possible to examine, in greater detail, the roles of TFIIB, TBP, and TFIIF30 as well as to identify the function of other basal transcription factors such as TFIIA, TFIIE, TFIIH, and TFIIJ. In addition, recent findings in our lab and others indicate that there are variations in the mechanism of basal examination of both the cis-acting elements in the promoters as well as the protein factors that are responsible for the promoter-specific effects that have been observed. These studies should lead to a better understanding of the fundamental transcription process and should eventually be applied to the molecular analysis of diseases involving the regulation of gene expression, such as AIDS and some forms of cancer. Identification and characterization of factors that are required for basal transcription. We have found that TFIIB, TBP, TFIIF30, and polymerase can accurately transcribe only a subset of promoters. It is thus, important to identify the factors that are required for transcription of the remaining genes. Examination of the molecular basis for distinct transcriptional properties of different promoters. We have been able to classify promoters into different functional categories based on their behaviour in various assays. The aim of these studies is to identify and to characterize the cis-acting sequences and the protein factors that are responsible for the promoter-specific effects. Kinetics and mechanism of transcription with a purified transcription system. We will carry out a systematic kinetic analysis of the assembly of the initiation complex with purified components. This work should provide new insights into the pathway leading to the assembly of a productive initiation complex. Analysis of transcriptional activation with the purified transcription system. A primary goal of these experiments is to determine the minimal components that are required to observe activation of transcription by promoter-and enhancer-binding factors.